PROJECT SUMMARY Clinical and epidemiological evidence suggests that ~40% of patients with ductal carcinoma in situ (DCIS) left untreated will progress to invasive breast cancer. However, due to our inability to distinguish lesions that will progress to invasive cancer from those that will remain non-invasive indefinitely, all DCIS patients are treated with surgery with or without radiation. This overtreatment dilemma demands the need for individualized patient care based on biomarkers that predict which patients will progress. Unfortunately, very little is known about how pre-invasive DCIS cells acquire the ability to invade the adjacent stroma, and how the stroma influences localized invasion has not been studied. The proposed studies aim to define the molecular interactions between infiltrating macrophages and pre-invasive epithelial cells, and uncover the mechanisms that promote tumor development in early stage lesions. Using unique preclinical models of early progression, we show that macrophages are recruited to pre-invasive lesions that have a high tumor-forming potential, polarized toward a pro-tumorigenic phenotype, and secrete the cytokine Gas6. We hypothesize that macrophages recruited to pre-invasive lesions induce cell survival and localized invasion in a Gas6-dependent manner. This hypothesis will be tested by the following aims: Aim 1: To define the oncogenic potential of Axl during mammary tumor initiation. Aim 2: To delineate the contribution of paracrine, autocrine and ligand-independent Axl signaling in early stage lesions. Aim 3: To decipher Axl transcriptional regulatory mechanisms, and identify Gas6- dependent tumor-promoting pathways. Our studies will utilize several mouse models, a unique xenograft model of DCIS progression, and a heterotypic 3-D culture system to dissect Gas6-mediated macrophage- epithelial crosstalk. Significance: The proposed studies will identify a mechanism by which DCIS cells proliferate and/or invade the basement membrane, and define how the stroma promotes progression to invasive cancer, addressing major knowledge gaps in the field of premalignancy. Recent studies suggest that the dissemination of cancer cells occurs as early as the premalignant stage, which begs the need to understand the mechanisms of cell migration and invasion in early stage lesions. Finally, Gas6 and Axl have been correlated with poor overall survival and therapy resistance in a number of cancers. Our findings will significantly advance the fields of DCIS biology and early stage breast cancer progression.